The dopamine transporter (DAT) is the plasmalemmal membrane protein that mediates the inactivation of released dopamine (DA) through its reuptake. DAT is the major molecular target responsible for the rewarding properties and abuse potential of amphetamine (AMPH), cocaine, arid related psychostimulants. AMPH, as a result of its ability to reverse the inward transport of DA causes DA efflux and therefore increases extracellular DA levels leading to the behavioral effects of AMPH. Recently, the molecular signals controlling AMPH-induced DA release have begun to be elucidated. Importantly, our group has shown that Ca2+/calmodulin dependent protein kinase II (CaMKII) is activated by AMPH and phosphorylates the DAT N-temninus leading to DAT mediated reverse transport of DA. We have recently developed the first detailed kinetic model describing the discrete steps in the functional cycle of the human dopamine transporter (hDAT;Erreger et al., J Neurosci 2008). Therefore, in Aim I we will use this model as a framework to evaluate which specific step(s) of the DAT cycle are affected by CaMKII and DAT N-terminal phosphorylation in order to support AMPH-induced DA efflux. Using animal model and pharmacological approaches it has been shown that D2 DA receptors (D2R) regulate the rewarding effects of AMPH. Importantly, our preliminary data and previous reports also show that D2R stimulation activates CaMKII. Therefore, in Aim II we will test the hypothesis in DA neurons that D2R signaling regulates AMPH-induced DA release in a CaMKII dependent manner. Changes in nutritional status (e.g. food-restriction, FR) regulate sensitivity to the rewarding and locomotor effects of AMPH. Notably, FR increases both D2R density and CaMKII activation. Therefore, in Aim III we will use acute striatal slices to determine how D2R signaling regulates AMPH-induced DA release in FR and control animals. In this Aim, we will clarify whether FR affects the ability of AMPH to cause DA efflux by altering D2R signaling. This proposal will allow the candidate to develop all of the skills and training to establish in his own lab and independent research program uncovering new cellular/pharmacological targets to regulate the actions of AMPH. PUBLIC HEALTH RELEVANCE: Abuse of psychostimulants such as amphetamine, methamphetamine, and cocaine represents a major public health problem. Treatment has been hampered by a lack of a complete understanding of how amphetamine works. By defining novel signaling pathways affected by amphetamine we will uncover new potential therapeutic targets for psychostimulant abuse.